The key to transformation of most organisms is the selectable marker gene, which allows one to identify and selectively grow transformed cells in the presence of a vast majority of untransformed cells. Unlike transformation of extensively studied organisms, such as Escherichia coli and Saccharomyces cerevisiae, transformation of many prokaryotic, and lower eukaryotic organisms is dependent on a limited number of selectable markers. Since transformation with a vector containing a marker gene normally results in the permanent loss of the selectable phenotype associated with that marker, it is difficult to proceed with further recombinant DNA-based modifications.
In organisms that are not genetically well-developed, the limited number of available marker genes can be a significant experimental handicap. Since the development of new markers requires considerable time and effort, it would be advantageous if methods existed to preserve or regenerate markers.
In some instances, a marker can be preserved by integrating markerless DNA fragments into the genome through cotransformation with an autonomously replicating, marker-bearing plasmid which is subsequently cured from the strain (Rudolf et al., 1985; Cregg et al., 1989). However, such a marker cannot be used for selective growth.
A method to regenerate markers has been described by Alani et al. (1987). This method employs an orotidine-5' phosphate decarboxylase gene (URA3) as marker and a powerful positive selection scheme that utilizes the drug, 5-fluro-orotic acid (Boeke et al., 1984), to select strains which have become Ura.sup.-, as a result of mitotic recombination events between repeated sequences placed on each side of URA3. However, this selection scheme requires a URA3-defective host organism which is not currently available in Pichia pastoris. To utilize markers presently available for Pichia, excision event products must be found by screening for loss of marker phenotype, a process which is likely to be tedious and may be fruitless, depending on the rate at which such events occur in Pichia. Consequently, a more exacting method, capable of high frequency regeneration of selectable gene markers, would be a major improvement over the present state of the art.